PROJECT SUMMARY This application is responsive to PA-15-313, Research to Advance Vaccine Safety (R21). Vaccinia virus (VACV) was used as a live vaccine for smallpox, a disease caused by variola virus. VACV has also been successfully used as a live viral vector for the development of effective human and animal vaccines, as well as immunotherapies and oncolytic virotherapies. However, VACV can cause complications in individuals with conditions such as atopic dermatitis, cardiac disease, and immunosuppression. Consequently, individuals with such conditions or with contacts that have these conditions are contraindicated for vaccination with replicating VACV vectors. We recently generated VACV vectors with a built-in safety mechanism that replicate only in the presence of tetracycline (TC) antibiotics (replication-inducible VACVs). In this system, a VACV gene essential for replication (eg, D6R) is inducibly expressed by TCs using elements of the tet operon. When administered as a vaccine (in the absence of antibiotics), the vector does not replicate but retains its immunogenicity, and therefore is safer for human use. Conveniently, the vector can be propagated in cell culture at high titers in the presence of TCs, unlike other replication-defective VACV-based vectors such as MVA. We also developed VACV vectors that replicate normally in the absence of antibiotics, but are replication-defective in the presence of TCs (replication-repressible VACVs). When administered as a vaccine (in the absence of antibiotics), the vector is replication competent like traditional VACV vectors (and therefore highly immunogenic), and treatment of any adverse reactions would be as simple as TC antibiotic therapy. We can further enhance the safety of our vectors by a fail-safe feature that links expression of the essential gene (D6R) with interferon-? (IFN-?), via an internal ribosome entry site (IRES). We showed that expression of IFN-? by VACV, either constitutively or inducibly, leads to complete attenuation of VACV in vivo, even when expressed at very low levels. Surprisingly, the virus is still able to grow to wild-type levels in vitro. Thus, both strategies can be combined to develop replication-defective VACVs inducibly or repressibly expressing D6R (under its natural promoter) and IFN-? (under a small murine IRES). The resulting vectors should be able to grow to high titers in vitro (thus allowing propagation) in the presence of TCs (inducible vectors) or absence of TCs (repressible vectors). Since expression of D6R will be linked to IFN-? expression, any potential replication-competent VACV that may originate during in vitro propagation or in vivo administration would be replication-incompetent in vivo due to concomitant expression of IFN-?. In Aim 1 we will determine the safety of replication-inducible and replication-repressible VACV vectors in vivo using immunodeficient SCID mice. In Aim 2, we will develop, characterize, and determine the safety of replication-defective VACV vectors with a fail-safe feature. We plan to use this VACV vaccine platform for rapid development of safe vaccines for infectious diseases such as Zika virus, immunotherapies (eg, personalized cancer vaccines), and oncolytic virotherapies.